Although stem cell/progenitor transplants promise unrivalled opportunities to repair damaged and aging nervous systems, success with this approach will only become possible after identification of the regulatory principles that control the functional differentiation of transplanted cells in the nervous system. Currently the technical approaches available to studying progenitor cell differentiation limit our ability to advance this field. Ideally one would repeatedly use non-invasive imaging approaches to whole animals to monitor the functional differentiation of transplanted cells. Unfortunately, available techniques such as MRI, that offer non-invasive imaging, do not provide the necessary spatial resolution. In this project we will develop a new approach in which we integrate two-photon imaging with two-photon photolysis to monitor and probe transplanted progenitor cell differentiation. To achieve this overall goal we have three objectives. First, we will refine optical stimulation and recording approaches that permit us to study the functional activity of transplanted progenitors. Using a genetically encoded Ca2+ indicator together with two-photon FRET microscopy and photolysis we will develop imaging methods that allow us to study their integration into host circuitry. Second, we will develop repeated in vivo imaging strategies that employ non-invasive two-photon imaging and photolysis to track the functional differentiation of transplanted progenitors in vivo for periods of up to one year. Finally, we will employ these imaging strategies to determine the relative merit of differentiating progenitors by exposure to growth factors either in vitro prior to or in vivo subsequent to transplantation. The development of vital imaging methods to assess progenitor cell differentiation will provide a new approach to allow the identification of the mechanisms which positively regulate differentiation and integration of transplanted progenitors. Since we will be able to assess outcomes based on cellular function that will surely be detected before behavioral consequences can be observed, the successful development of this imaging approach will accelerate the pace of research with behavioral outcomes goals.